Recovery of rare sugars



Patented July 10, 1945 I RECOVER-YUP Louis Laufer and Jesse Charney,NewYoi-k; N. Y .1

assignors to Schwarz Laboratories,

Inc., New

Y NoDrawin'gJ ApplicationAugust14519 12, r

a 'Serial No.454,838 t zsoiaim (creed-+209) Our present inventionisdirected to an improvement upon that ofour'prior application, Sej rialNo. 454,022, filed August 7, l942g which is concerned with theextraction of d-ribose and-"kinentremaining th used asttheristartingmaterial. i l a a in l The treatment set forth has itspreferredapplication to the extraction ofKd-ribose 'fro'msubstancescontaining ribonucleotides that includes ad'enylicacids,

some or all of Xanthylic,inosinic,guanylic and According to theinvention, nucleic acid may .be freed in" crude form from yeast cells orother dred' sugars from biologic substancesthat bear 5 biologic sourceand thenbroken up intoiitsMconpurinenucleotides or nucleosides. h l 3 ll stituentnucleotides,or alternativelythenucleic An object of thepresentinventionis to recover acidoro'ther'nucleotidebearingsubstance'may be} 1 by a relatively simple process a high yieldofj freed from the yeast cells for the-like, and concur-f d-ribosedirectlyfrorn nucleic acid bearing subrently bebroken up intosuchconstituentjnuclo i stances, though the nucleicacid be recoveredbytides; frorn which nucleotidesthe rar e sugar: is

the use of iron v chloride and have substantial splitoflin marineralsotaught the presenting amounts of ferric or other impurities therein;the vention. Each of the alternative: procedures inpresent inventionserving toeliminate ontheone 'volves a series of steps which includes amild hyhandthe need for first freeing of ferric or other n dialysis, andthesewillnoiv be set f orth ingreater reagent the nucleicacid thatservesas thesource detail. v By mild hydrolysis is meant treatment ofsugar, and on the other; theneed for large with dilute: reagents at orbelow boiling point at quantities of costly precipitating reagentsandf'or atmospheric p e 1 elaborate processing, V According to the firstof said alternative pro- The presence of iron in the nucleic acid usedcedures for recovering nucleotides; crude nucleic as the startingmaterial for the recovery of acid is first prepared as follows: Theprocessed d-ribose or other pentose would lead to loss by yeast-"orother source of purine nucleotides aiter oxidation of much of the sugarin the course of treatment withalkali to free'the-nucleicacid fromreleasing nucleosides by prolonged alkaline treatthecells is treatedwith mineral acid to bring the ment under pressure and heat resorted toin the (pl-I to a value close to the isoelectric point of processofsai'd prior application. ii f26 much of the proteinmatter.Uporrboiling, such While iron-free nucleic-acid useful as a source 1 potein i p c p t te a 't n te d l h of d-ribosemay be derived frompressed yeast or thefnucleic acid content remainsfinsolution. The othersource of nucleic acid by extractionfollowed filtrate W ju e y theaddition hydroby precipitation with alcohol accordingtofam'ilchloricacidto Isl-14.5 and then boiled again, iar practice, suchprocedure is costly inmaterials. 30 whi h results th p p n o mo e oft eWhile the high cost of materials isavoide'd by proteinmatter. Afterfilteringm the pH of :the the use of iron chloride to precipitate crudenu- 'filtrateis'furtheradjusted by theaddition of more clei-c acid fromthe biologic material bearing the hydrochloric acid, to -a pH-betw eenlj and 15, same, according to a, recently developed process, desirablyto about2. 5. Approx mately: 1 per cent considerable expense [isinvolved in processing by weight ofiron chloride; preferably ferricchlsuch nucleic acid for removal of all irontherea ride, is then added,yieldingthe desired precipifrom with a view to obviating the loss ofsugars tate-off crudenucleic acid, which after washing, by oxidation asabove pointedout. serves as-thesource of the d-ribose;

According to the invention, the prolonged alkaa Byfith st ps e for h forproducingthe crude line treatment of the nucleic acid under pressure 40nuclelc'acid; not'fipnly is muchjofjthe protein inatand heat isdispensed with, and the pentose issplit ter removed, but the final"adjustment of p the pH off from the nucleotides withoutfirstj convertingI p ec udes e precipitation of man o there- 1 them 'to nucleosides. Thisis accomplished by a puri ies, when the f erricchloiide is acidhydrolysis after a short alkali treatment at subsequently added,For'thatreason the precipiatmospheric pressure and relatively lowertemperiate jha a c entlo si ieh s 7 03 D 081 ature; by which acidhydrolysis not only the oijpure nucleicacid an'd the impuritiesare suchpentose, but thephosphoricacid as well are split S HOfi 0 t r th h seara ion O alrich off from the nucleotides. A high yield of the rareyield 01 pe tose thererromby the steps to be set sugar is garnered incarrying out this procedure, forthihereinafter. l i i even though crudenucleic acid withferric' re- '50 "The" nucleic acid rega'rdless'Whetherit be in crude or contaminated form a above described,

orfvvhether it be previously refined,.;is subjected t'o'fniild'alkalinehydrolysis? Fortl'ii's plirposeQthe crudejprjrefined"nucleicjacid ispreferably'boiled v in caustic soda, 1 desirably 5" per cent sodium by adroxide for a period of 30 to 45 minutes, and the precipitated gums,iron hydroxide and impurities are filtered off. By that treatment theacid is broken down into its constituent nucleotides, for instance, thepurine nucleotides, adenylic and guanylic acids, and the pyrimidinenucleotides, ytidy and uri li ac s According to the; second of thealternative procedures above referred to, the yeast or other source ofnucleotides is boiled for about on hour with alkali such as sodiumhydroxide in a 3 to per cent, preferably a 5 per cent aqueous'jsolution,

in conjunction with calcium compound such as lime or equivalent alkaliearthcompoun phosphates extraneous of the nucleotides," are now filteredout with the other?yeastsolida-leav- @The If resultant precipitatedimpurities which nclude,

ing a filtrate that contains in solution the'free nucleotides of thehydrolyzed nucleic acid, such,

as those above mentioned, that are contained in theye'ast orthe like.-ffj '1 i Whether the nucleic acid is broken; down into its constituentnucleotides :only after it has been released from the yeastcells by thefirst of the foregoing procedures, oris thus released and broken downconcurrently in-a single step, such the latter step by adding cuprousoxide and sulphuric acid until the final pH of the solution is from 2 to3.5. p

The salts are filtered out and washed with plain water, either hot orcold. The safeguard of using sulphite solution for the wash isunnecessary, as the nucleotides, do not tend to oxidize as readily asthe 'nucleosides. In: this operation, the great bulk of the pyrimidineswill remain in the filtrate. The copper salt is now suspended insulphuric acid solution of 0.5 to 2.0 normal strength and hydrolyzed atboiling temperature for a substantialfperiod of two to four hours, whichresults in splitting off from the nucleotides the phosphoric acid andthe pentose. purinesalts, comprising copper adenine andcop per guanineare now filtered ofi".

The insoluble cuprous The pI-I of the filtrate is adjusted to pH 6.5 to8.5 by the addition of oxides or hydroxides of alkaline earth metals,desirably barium hydroxide or lime, which precipitates barium or calciumphosphate and sulphate, cuprous and cupric'hy- The filtrate after beingthus substantially neu- .tralized is then treated with copper cation anddesirably oxide of sulphur anion,,of which only oneis in the reducingform, to provide cuprous ions-for yielding a pr'ecipitateof cuprousnucleotidessuch as cuprous adenylic acid and cuprous guanylic acid whichare new chemical compounds, and are claimed as such. The pyrimidinenucleotides remainlargely in solution.

, The precipitate is thereupon subjected to hydrolysis with sulphuricacid, which ,results in splitting off both the glycosidic and the phos--.phoric ester linkage of the nucleotides,leaving in suspension insolublecuprous purines such as cop- ;per adenine and copper guanine, andbringing ,intosolution free pentose and free phosphoric acid. Theimpurities are removed from thefil- 'tratefby precipitation withalkaline earth hydroxide, leaving substantially pure d-ribose inolution. I a I The treatment of the nucleotides with cuprous ionresembles that specifiedffor treatment of the nucleosides in the earlierapplication. As is the "case with said nucleosides, those purinenucleotides which form insoluble salts or that have an unsubstituted No.7 position in the purine group are more or less selectivelyprecipitated, the other nucleotides remaining insolution;

Preferred controls for recovering the d-ribose from the purinenucleotides in solution, regardless by which of the alternativeprocesses such solutionis prepared will now be set forth:

The filtrate is first treated to form insoluble copper salts of thepurinenuc'le'otides. is accomplished by first neutralizing the filtrate,desirably with sulphuric acidand thenv adding copper sulphate and sodiucid s'ulphite until a final pH of between '2.0" an d,8."5,' butpreferably jtrali'ze'd, preferably with sulphuric acid to a pH of about8.5. To this solution is added 310 c. c. ,of a solutionma'deof 250 c, c.of copper sulphate solution (25 grams CuSO4.5l-I2O in 100 c. c. of

a In a preferred process droxide, and organic impurities, Th d-riboseremains in the filtrate together with small amounts ofimpurities. I

for: concentrating and purifying the pentose v separated as abovebriefly set forth, the same, after concentrationis absorbed upon asuitable inert material of large surface capacity such aspurifieddiatomaceous earth with which a quantity of activated carbon isadmixed. The mass is subsequently dried at reduced pressure andextracted by means of a soxhlet or similar apparatus with suitablesolvent, such as 3.5 to -5 partsby volume of ethyl ether and one part of95 per cent ethyl alcohol that selectively removes only the pentose andleaves the impurities as a residue in the inert mass. Uponevaporationlof the solvent and seeding, thepure pentose is obtained incrystallized form. V i v H Specifications in considerable detail forderiving refined d-ribose from nucleic acid according to the inventionwill now be; set forth, to assure technical compliance with thestrictest interpretation of the statutory requirements;

50' grams of nucleic acid which may be derived from the pressed yeast orother source thereof by the procedure above described and which may. be

approximately 85 per cent pure is introduced in 2 00 c. c. of a 5 percent sodium hydroxide-solubined with the filtrate.

The filtrate which may be about 300 c. c. is diluted with" water toabout 1000 c. c. and neuwater), and 60 c. c. of sodium acid sulphitesolution, ,(210'gr'ams Nal-ISOa in 750 c. ,c. of water). reduces the pHto about 4.0. This step is desirably carried out at between 25 degreesand '100 degreesyC. An immediate precipitationoc- :cur s, which isessentially of cuprous salts of .adenylic and guanylic acids.

The precipitate is separated out by a centrifuge and then washed byings, each with'about 750 c; c. of water are between 3.0 teasisreachedor, alternatively t9 lsufiicient The liquid contains most ofthe pyrimidine nucleotides; The'washed precipitate consists of thecuprous salts of all of the purine nucleotides and some pyrimidinenucleotide.

The washedncuprous nucleotides are dissolved Buchner funnel, and Washedwithwater toa total filtrate volume of 1l00.to1200 c. c., leavingrelatively pure cuprous adenine and cuprous-guanine salts asthe-residues in l i t I To the cooled filtrate is added a slurry ofBa(OI-I)2until the pH of the mixture is about 7;

Substantially all of -lthe sulphates, phosphates, copper and organicimpurities are thereby-precipitated, leaving in solution the d-ribo sewith but small. amounts of impurities. The slurry is filtered andwashed: with aboutxtw-o liters of water. i l i The filtrate of about3500 c. c. is now concentrated under reduced pressure to a syrup of 10to c. c. which is thereupon diluted with Waterto30 c. c. andfilteredthrough a gooch crucible with washing to: a total .filteredvolume of 125 c. c. This filtrate is treated with two grams of carbonatabout '70 degrees C. for 15 minutes, filteredvand washed to a volumeof 200 c. c. after which it is concentratedatreduced pressure to to c.c., a ksyrupycontaining about, 50 per cent solids. l a a The25 to 30-0.c. of relatively pure d ribose solution thus prepared is poured on 30grams of purified diatomaceous earth, one commercial form of which thatis particularly suitable, being identified as C'elitei 503 r to which isadded 5 to 10 per cent ofactivated carbon.- Themass is dried at 40degrees C. in a vacuumdesiccator. The dried material is thereuponextractedfor four hours in a soxhlet. apparatus with a solventconsisting approximately fouriparts of ethyl ether by volume and onepart of 95 per cent ethyl alcohol by volume. .Thetextracted sugarcrystallizes out when the solvent is evaporated off and the resultantsyrup seeded. The yield is about 8 grams of d-ribose with a meltingpoint of 85 to 87 degrees C. I l

While the most economical,- and industrially the most advantageousprocedure is to derive the sugar directly from substances bearingnucleotides or from crude nucleic acid as above set forth it will beunderstood that the process in certain of its aspects may,withinthescope of the invention, be performed with refined nucleic acidas the starting material. j n

It will be understoodthat. the procedure above setforth may be appliednot only for the preparation of pure d-ribose but of other pentoses ofthe typefound incorporated in thenine position in the purine group ofnucleotides. Among such sugars are rhamnose, xylose, arabinose anddesox'yribose. Among thetypes of nucleotides tainediinwthe abovedescription shall be interpreted as illustrative and not in a limitingsense.

Having thus described ourinvention, whatwe claim asnew anddesire tosecure by Letters Pat ent of thesUnited States is:} it

1. The process which consists in subjecting solutions oinucleotidebearing substanceto mild alkaline hydrolysis, then treatingflthe samewith i a copper compound and an acid that conjointly aiiord cuprous ionswith resultant precipitation oi' substantially insoluble cuprous saltsof nuleotides andthen separating and washing said precipitate. y n v p n2. The process which consists in treating solutionscontainingnucleotides with cuprous ions in anacid medium with theresultantselective separation'in the form ofa precipitate ofthosenucleotides which formdnsoluble cuprous salts. a.

3. The process which consistsin subjecting nucleotide bearing substancesto mildfalkaline hydrolysis and then treating the same with cuprousionsin an acid medium with theresult-l ant selective separation in the formof a ms:

cipitate oi -thos purine nucleotides present that i have anunsubstituted N o. '7 position in the purine 4; The process of splittingcomplex organic molecules in biologiccell tissue substantially toisolate the purine nucleotides thereof from the pyrimidinenucleotidesthereof, by subjecting the complex to mild alkalinehydrolysis and then treating the solution with cuprous ion in thepresence'of an acid with the consequent precipi-f tation of cuproussalts of the purine nucleotides while the pyrimidine elements remainlargely in solution. i

5. The process which consists in splitting complex organic molecules ofbiologic cells to separate the purine nucleotides from the pyrimidinenucleotides, by subjecting the complex to mild alkaline hydrolysis,filtering, then treating the filtrate with cuprous ions in an acidmedium with the resultant selective separation in the form of aprecipitate of the cuprous salts of those purine nucleotides that havean unsubstituted No. 7 position in the purine group, while thepyrimidines and substances bearing thesame arenot only nu- A Y cleicacids, but such acids as} thyme-nucleic acid,

inosinic'acid, xanthylic acidand likesubstances that may serve as thesource of rarepentoses according tothe present invention;

could be made Without; departing from; the scope and derivatives thereofremain largely in solution.

from like nucleotides with substituted No. 7 position in the purinegroup which consists in treatcrude nucleotides with a copper compoundand an acid which conjointly afford cuprous ions that combine withsaidnucleotides, isolating those of l the cuprous salts thus formedwhich are insole uble and splitting both the sugar and th phosphoricacid therefrom by acid hydrolysisaw 8. The process comprising thetreatment of crude purinenucleotideswith a copper compound and an acidwhich conjointly affordcuprous ions that selectively combine chemicallywith purine nucleotides that: have an unsubstituted No. 7 p0 l i sitionin the purine group, isolating the cuprous 1 salts thusformed which areinsoluble andsplitting both the sugarsand the phosphoric acid'ofltherclaims, itxis intended that all matter coni 6. The process ofseparating nucleotides with unsubstituted No. \7 position in the purinegroup V 9. Th process for preparing r'eflnedxpentoses from crude purinenucleotides which consists in treating such nucleotides. with cuprousions to form insoluble cuprous saltsofthe said :nucleotides, separatingthe. precipitate .oiisuch: salts, suspending the same :in an acidm'ediumlat tem-.- perature near the boiling point. andhydrolyzing thesame, to separate out insoluble cuprous. salts of'purine, removingthesame, and. separating, refining and crystallizing th free pentosefrom thefiltrate.

10. The process of preparing pentoses compris-v ing the treatment ofsolutions.. bearing purine nucleotides at pH 2.0 to 8.5 with a copper.compound nand an acid which coniointly aiiord cuprous ions, thereby toisolate th insoluble cuprous salts of said nucleotides washing saidsaltsinwater and recovering the sugars by hydrolysisvin anacid mediums,;1 1,."The processoi. preparing d-ribose-irom substance containingribonncleotides that include some or all of xanthylic, inosinic, guanylio and adenylic acids, which consists in treatingsolutions containingsaid rlbonucleotides with cuprous salts and sulphuric. acid at pl- Ibelow. 6,0 at 2 5 to .0.0.. degr e -0., fo l wed Wheelin h by. iprecipitate cuprous purine ribonucleotides, washing, such salts toremove impuritiesand splitting ofi the d-ribose from such substantiallypure salts.

,12, The process of .prep'aring raresugars comprising the treatment ofsolutions. ,,of purine nucleotide bearing substances atpl- :Z-QztQ withcopper cation and oxide of ,sulphuranion of which. only one is in.thereducing forrn at 25 degrees to 100 degrees C., isolating the resultant precipitate of insoluble cuproussalts of nucleotides thusformed, washing the salts with water, hydrolyzing the said. salts indilute acid by boiling from two to four hours, removing the resultantprecipitate of insoluble. cuprous salt of fpurinesand recovering, the.sugar from the filtrate. r

13. The process of preparing sugarsfrom ,solutions containing sugarscombined with purines which consists in treating the sugar bearingsubstance with copper sulphate solution in ,con-

junction with solutions affording sulphurous anions under conditions ofacidity which render the pH of the treated solution between 3.0 and 6.5,thereby to .isolate cuprous salts. of purine nucleotides, washing saidsalts,,hydrolyzing the same in approximatel one normalsulphuric acid. byboiling'for two to four hours; removing therefrom the insoluble cuprouspurines thus precipitated, adjustingthe filtrate to pH 6.0 to 8.5 with amaterial selected from thetgroupcon sisting of the oxides and hydroxidesof the alkaline earth metals, removing the precipitate so formed andrecovering substantially pure sugars from the filtrate. 14. The processof preparing sugars from solutions containing phosphoric sugar esterscom bined with purines which consists in adding cuprous salts andsulphuric acid thereto, there by to precipitate insoluble cuprous purinenucleotides, washing such salts, hydrclyzing the substantially purecuprous nucleotides thus obtained in one normal sulphuric acid atboiling temperature for two to four hours, followedby the removal of theinsoluble cuprous purines therefrom, adjusting the filtrate to pH 6.0110-85 with a material selectedfrom the group consisting of the oxidesandhydroxidesof the'alkaline earth metals, removing the precipitates.formed and recovering substantially pure sugars from solution, removinginsoluble cuprous salts 'of' guanine and adenine therefrom, neutralizingthe filtered hydrolyzate with a material selected from the groupconsisting of barium and calcium oxide and hydroxide of pH 6.5 to 8.5 toremove residual copper phosphate and'sulphate and impurities, filtering,further purifying with activated carbon and recovering substantially thepure pentose from the filtrate. i

16. The process of preparing d-ribose from purine ribonucleotideswhich-consists of treats ing ribonucleotide solution with coppersulphate solution in the presence of sulphurous acid to yield a solutionof pH 3.0 to 6.5, thereby to precipitate cuprous purine ribonucleotides,washing said salts to remove impurities, hydrolyzing the washed salts byboiling from two to four hours in approximately normal sulphuric acidsolution, removing the insoluble cuprous purines thus formed,neutralizing the filtered hydrolyzate with a substance selected from thegroup consisting of barium and calcium oxide and hydroxide to pH 6.5 to8.5 to remove residual copper sulphate, phosphate, sulphite and organicimpurities, filtering, further purifying the filtrate with carbon andrecovering substantially pure d-ribose from the filtrate. 17. Theprocess of recovering pure crystalline d-ribose from substantially pured-ribosesolutions, which consists in decolorizing and concentrating thesolution, absorbing the syrup thus formed in an inert'material bearing 5to 10 per cent of activated carbon, drying the mass, extracting theresulting dry material with selective d-ribose solvent of low boilingpoint and inert to the impurities present, evaporatin the solventextract, and seeding and crystallizing pure d-ribose from the syrup thusformed.

18. The process recited in claim 17 wherein purified diatomaceous earthserves as the inert material for absorption of the d-ribose syrup.

19. The process recited in claim 17 wherein the extracting solventcomprises about four parts by volume of ethyl ether and one part byvolume of per cent ethyl alcohol.

20. The process of deriving pentose from nucleotide bearing cell's,which'consists'in freeing the nucleic acid from the cells and breakingthe same down into its constituent nucleotides, and then performing thesequence ofsteps which consists of a mild hydrolysis of the nucleotidesin solution at boiling temperature, precipitating from the filtrate thepurine nucleotides in the form of cuprous salts thereof by theadditionof cuprous ions in an acid medium, hydrolyzing the precipitatein sulphuric acid and thereby leaving a residue of cuprous salts ofpurines in a solution of pentose and phosphoric acid and adding alkalineearth hydroxide to the filtrate'to precipitate out the phosphoric acidand other impurities, yielding a filtrate comprising substantially purepentose solution.

21. The process set forth in claim 20 in which preparing pentoses whichfor the purpose of freeing the nucleic acid from the nucleotide bearingcellsand breaking it down into its constituent nucleotides, the nucleicacid 3 solution is adjusted in pH and boiled to precipitate much of theproteinmatter, the filtrate is then readjusted in pH to approximately4.5 and boiled again and after filtering reduced in pH to between 1.5and 4.5, and thereupon iron chloride is added, and 'in which the mildhydrolysis is in alkali solution.

22. The processrecited in claim 20 in which the nucleotide bearing cellsare treated with alductedunderfinalpH of2to 8.5. i n

-2o. The combination recited in claim 23 in i which the acid hydrolysisof thecopper salts is i conducted in sulphuric acid in solution of 1 to2 kali in 3 to 10 per cent solution together with alkaline earth salts,for about one hour for the mild hydrolysis set forth. t 23. The processof producing substantially pure d-ribose from nucleotide bearingbiologic cells which consists in separating the nucleic acid from thecells by caustic treatment, filtering it,

precipitating out proteinmatter under controlled pH, thereupon reducingthe pH to about 2.5, adding 1 per centof iron chloride, washing theprecipitate thus obtained, subjecting the latter to mild alkalinehydrolysis, and after filtration thereof precipitating the purinenucleotidestherefrom by the addition of cuprous compounds in the presence of acid and hydrolyzing the water-washed precipitate in diluteboiling sulphuric acid, thereby to precipitate the purines and to splitoff dribose and phosphoric acid and finally precipitating out the otherimpurities with alkaline earth hydroxide, yielding a solution ofsubstantially pure d-ribose. J l i 24. The combination recited in claim23 in which the mild alkaline hydrolysis isconducted with 5 per centsodiumhydroxide boiled for tominutes. t 1

25. The combination recited in claim 23 in which the precipitation withcuprous salts is connormal strength by boiling for two to four hours.

27. The combination recited in claim 23 in which the mild alkalinehydrolysis is conducted in 5 per cent sodium hydroxide by boilingtherein for 30 to 45 minutes, the precipitation of cuprous salts isconducted at a pH of from 3 to 6.5 and the hydrolysis of the coppersalts is conducted;

in sulphuric acid solution of one to two normal strengthand boiled fortwo to four; hours.

28. A composition consisting of a mixture of substantially pure cuproussalts of purine nucleotides that have an unsubstituted No. 7 position inthepurine group. l r

29. A composition consisting of a mixture of substantially pure cuproussalts of g'uanylic and adenylic acid. t LOUIS LAUFER;

JESSE CHARNEY.

